It is generally accepted that elevated blood cholesterol is a causative factor of coronary heart disease. Moreover, it is recognized that high blood levels of the form of cholesterol known as low-density lipoprotein (LDL) can contribute to cardiovascular disease. Guidelines have been established to indicate to doctors when patients should be considered at risk. Desirable is less than 200 mg/dl, borderline is 200 to 239 and high is greater than 140 mg/dl Schueker et al., Arch. Inter. Med. (1991) 151:666-673. If total cholesterol is greater than 240 mg/dl and/or LDH is above 160 mg/dl, therapeutic treatment may be needed. [See Goodman, Amer. J. Med., 90:2A-32S to 2A-35S (1991).]
The development of the present invention began with experiments conducted at Iowa State University, Ames, Iowa, U.S.A. in which metabolic products of leucine were feed to domestic animals. As described in U.S. Pat. No. 4,760,090 of Steven L. Nissen, it was found that ketoisocaproic acid (KIC) can be feed to cattle and sheep for enhancement of growth and feed efficiency. It was observed that during such KIC feeding there was some reduction in plasma cholesterol, and also in the deposit of cholesterol in the meat. (See 4,760,090, col. 5-6, Table B.)
In another application of KIC feeding, egg production of laying chickens was increased, as described in U.S. Pat. No. 4,764,531 of Steven L. Nissen. It was found that the eggs of KIC feed chickens had reduced yolk cholesterol (4,764,531, col. 4, Table B).
In later experiments carried out by Dr. Steven L. Nissen at Iowa State University, .beta.-hydroxy-.beta.-methylbutyric acid (HMB) was fed to domestic animals. The effects obtained were different than with KIC. Metabolically, KIC and HMB are not equivalents. KIC is the only metabolic product of leucine, while HMB is a minor product of KIC metabolism.
Leucine is either used for protein synthesis in the body or is converted directly to KIC. In the mitochondria KIC is decarboxylated to isovalarylCoA and then further metabolized to ketone bodies. In certain disease conditions, such as isovalaric acidemia, an alternate oxidative pathway for KIC has been observed, which appears to produce .beta.-hydroxy-.beta.-methylbutyrate (HMB). In atypical cases, such as a genetic absence of the dehydrogenase enzyme, there is evidence that HMB can accumulate in the urine: Tanaka, et al. Biochim. Biosphys. Acta. 152:638-641 (1968). Also, in acidosis conditions, HMB levels can be increased in urine: Landass, Clin. Chim. Acta. 64:143-154 (1975). This presumably occurs by oxidation of KIC to HMB by the enzyme .alpha.ketoiscaproate oxygenase (Sabourin, Metabolism (1983) 32:160-164). Increased urine HMB can also occur in cases of biotin deficiency (Mock, J. Lab. Clin. Med. (1988) 240-247). The only evidence for normal HMB production is in lambs and pigs, Vankowering and Nissen, Am. J. Physiol. (1992) 262:E27-E31. In this study it was estimated that &lt;10% of leucine metabolic is via HMB production.
The differing activities of HMB as fed to domestic animals provided the basis for additional patents of Steven L. Nissen. His U.S. Pat. No. 4,992,470 discloses the administration of HMB for enhancing the immune response of mammals and as an ingredient in the raising of meat producing animals (e.g. ruminants and poultry) to increase lean tissue development. (See U.S. Pat. Nos., 5,087,472 and 5,028,440 of Steven L. Nissen.)
There has been a scientific effort to determine how cholesterol is synthesized in the bodies of mammals. It was known that acetate can be synthesized into cholesterol. Research investigations in the 1940's and 1950's concentrated on experiments with organic acids which also incorporated acetate and whose tracers could be incorporated into cholesterol. A small group or organic acids appeared to meet these qualifications. This included .beta.-hydroxy-.beta.-methylglutarate (HMG), .beta.-hydroxy-.beta.-methylbutyrate (3-hydroxy isovalarate), .beta.-.beta.-dimethylacrylate (DMA), isovalarate, and .beta.-methyl-gluconate (.beta.MG). 14C from 14C-acetate can be detected in all these compounds. Today it is thought that HMG-COA is the obligatory precursor to cholesterol, and the other compounds referred to herein are somehow incorporated in cholesterol by interconversion with HMG. (Adamson et al. 1957, Biochem. Biophys. Acta, 23: 472-479.) Thus, although there is a biochemical relationship between HMG and HMB, it is not clear if there is any relationship between the compounds regarding effects on cholesterol metabolism.
Experiments demonstrated that feeding HMG to rats could decrease total serum cholesterol by up to 20%. Effects on LDL cholesterol were not reported (Yousufzai et al., Lipids, 11:526-529).
Only limited human studies have been carried out with HMG. One study did measure the effect of HMB on subjects with familial hypercholesterolemia, and LDL was measured. A modest decrease in total cholesterol and LDL cholesterol was reported. (Lupien et al., J. Clin. Pharm., 19:120-126, 1979.)
After 8 weeks of being fed 3 grams of HMG daily, total cholesterol decreased from 404 to 353 mg % (-13%) and LDL decreased from 333 to 307 mg % (-8%). HDL cholesterol decreased approximately 35%. Thus, HMB appears to act differently from HMG in humans in that the effect is more pronounced and results in a specific decrease in LDL cholesterol but not in HDL cholesterol.
U.S. Pat. No. 3,629,449 claims that oral HMG can reduce serum cholesterol (total) and blood lipids (triglycerides) in warm-blooded animals.
Only one study is known where HMB was fed to animals, and an index of cholesterol metabolism measured: Gey et al., Helvetica Chim. Acta, 40:2354-2368 (1957). In that study HMB was fed to rats at a rate of 0.5 g/kg body weight for 2 and 4 days. At the end of the study, cholesterol synthesis was measured by removing the liver which was incubated in slices with 14C acetate. Cholesterol was isolated following the incubation and radioactivity quantitated. It was found that HMB had not significantly lowered the rate of acetate incorporation into cholesterol by the rat liver as compared to controls. In the same paper, an in vitro interaction of HMB and acetate incorporation was assessed. When HMB was added to the media at very high concentrations, it was found that there was no significant inhibition of acetate incorporation compared to the control values.